Section on Physical Biochemistry, OD/NICHD conducts experimental and theoretical studies of structure and function of biomolecules. In recent years we invested most of our efforts into investigation of molecular mechanisms of Osteogenesis Imperfecta (OI) and fundamental studies of underlying processes of type I collagen folding, interactions and fiber assembly. The primary goal of these studies is to gain better knowledge and develop novel techniques for diagnostics, characterization and treatment of OI and other bone and connective tissue disorders. Over the years we found that collagen triple helix is intrinsically unstable at physiological conditions. Micro-unfolding of its most thermally labile regions accompanies fiber formation. In fibers, collagen helices are protected from complete unfolding but they constantly undergo transient local unfolding and refolding contributing to the unique combination of elasticity and strength of connective tissues. Formation, hydration, mechanical properties and the extent of molecular micro-unfolding in fibers depend on the balance of forces between collagen triple helices. We reported first direct measurements of these forces and established their physical nature. OI mutations affect collagen folding, interactions and fibril formation and properties through a variety of different mechanisms. To better understand these mechanisms, we continue systematic studies of mutant collagens with substitutions of obligatory glycine residues (responsible for over 80% of moderate to lethal OI cases) as well as studies of interesting and unusual individual OI cases. In the last year, in collaboration with BEMB/NICHD researchers we described changes in thermal stability, structure and enzymatic processing of type I collagen from a patient with a combination of OI and Ehlers-Danlos-Syndrome (EDS) symptoms resulting from an atypical point mutation (Arg888Cys in alpha1(I) chain) not involving any of the obligatory glycine residues. We expanded our analysis of thermal stability of glycine mutants from the collection of BEMB patients. The latter study revealed that the position of a mutation within a certain domain is more important than the identity of the substituting residue or its immediate local environment. From the data for over 30 mutations we deduced that collagen triple helix contains at least six different structural domains. We completed mapping of the N-anchor domain formed by 85 amino acids at the N-terminal end of the helix, characterized structural changes caused by mutations within this region and proposed a molecular mechanism explaining the appearance of a distinct OI/EDS phenotype in patients with these mutations. We found that two other domains coincide with regions of increased incidence of lethal OI in the newly compiled database of over 800 mutations and hypothesized that the domain structure of the triple helix is an important factor contributing to the genotype-phenotype relationship. Although abnormal procollagen folding and stability are at least partially responsible for pathology, we are also finding examples of OI mutations affecting the phenotype through changes in type I collagen secretion (e.g., in the Brtrl mouse model), by directly altering collagen-collagen interactions in fibers (e.g., in the oim mouse model) and likely through other mechanisms as well. The best understanding of these mechanisms can be achieved through murine models, as in the two examples above. Thus, one of the most exciting and promising developments of the last year was initiation of a new collaboration with the University of Maryland Medical School to study a novel OI mouse model with Gly610Cys substitution knocked into col1a2. By utilizing a set-up for high resolution micro-infra-red spectroscopy recently developed in our group, we found that the amount, structure and alignment of mineral crystals in femurs from Gly610Cys animals appear to be within the normal range. Instead, abnormal mechanical properties of bones in mutant animals appear to be related to altered structure and mechanical properties of collagen fibers. Our measurements showed that mutant collagen molecules have significantly lowered thermal stability (by ~ 4 oC). Nevertheless, they are effectively folded, secreted and incorporated into fibers. We are currently investigating potential molecular mechanisms of their effects on fiber properties. Another important direction of our research is closely related recognition and assembly reactions involving DNA, which play an important role in packaging of genetic material inside cells and viruses and in many other fundamentally important biological processes. In particular, we uncovered several common physical principles of interactions which govern formation, structure and physical properties of collagen and DNA aggregates. Our theory of these interactions provided explanations for the observed counter-ion specificity of DNA condensation, DNA overwinding from 10.5 base pairs (bp) per helical turn in solution to 10.0 bp/turn in aggregates, nontrivial cholesteric pitch behavior upon compression of DNA aggregates, subsequent transition from the cholesteric to hexagonal (hexatic) phase, and multiple quasi-crystalline phases of even more densely packed DNA aggregates. It also suggested that electrostatic interactions might contribute to sequence homology recognition and pairing of intact DNA double helices observed prior to genetic recombination. One of the most important and yet controversial predictions of this theory, distinguishing it from other models, was that strong azimuthally-dependent interaction should align strands and grooves on opposing surfaces of adjacent molecules. Such alignment was observed in crystals of DNA and nucleosomal particles, but in contrast to our predictions it was not traditionally expected for highly hydrated, liquid-crystalline aggregates. As proposed by Franklin and Gosling, it was expected that in such aggregates DNA should be ?relatively free from the influence of neighboring molecules, each unit being shielded by a sheath of water?. The latter assumption was used, e.g., by Watson and Crick, Wilkins et al., and Franklin and Gosling for interpretation of DNA diffraction patterns in their celebrated set of back-to-back papers on DNA structure. Thus, within the last year we revisited these classical patterns using a more detailed set reported by Zimmerman and Pheiffer in 1979. We adapted the classical Cochran-Crick-Vand diffraction theory to account for possible short-range azimuthal order in the aggregates and analyzed the observed changes in the diffraction patterns caused by varying aggregate hydration. We found that the observed changes do not affect the classical interpretation of DNA structure. However, they unequivocally reveal strong azimuthally-dependent interactions between adjacent molecules up to ~ 20 ? surface-to-surface separations in good qualitative and quantitative agreement with our predictions, lending strong support to our theory.